METTL5 serves as a diagnostic and prognostic biomarker in hepatocellular carcinoma by influencing the immune microenvironment

Despite the abnormal expression of 18S rRNA m6A methyltransferase METTL5 being reported in some types of human malignancies, but its effect on hepatocellular carcinoma (HCC) remains to be unclear. This study aims to elucidate the influences of METTL5 on the carcinogenesis and progression of HCC. Expressions of METTL5 gene, transcript, protein, and promoter methylation in HCC were examined through multiple databases, c-BioPortal was used to confirm the genomic alterations of METTL5, the biological functions, target networks of kinases and microRNAs of METTL5, and its interactive differential genes were investigated through LinkedOmics. The possible correlation of METTL5 with the tumor-related infiltration of immune cells for HCC were explored comprehensively by using the online tools of TIMER and TISIDB. Expressions of METTL5 gene, mRNA, and protein were considerably overexpressed in HCC samples in comparison with healthy samples. The high methylation of the METTL5 promoter was observed in HCC tissues. Elevated METTL5 expression exhibited unfavorable survival outcomes in HCC patients. METTL5 expression were enriched in the signaling pathways of ribosome and oxidative phosphorylation, mismatch repair, and spliceosome through the involvement of several cancer-related kinases and miRNAs. The METTL5 expression has a positive correlation with the infiltration degree of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in HCC. Marker genes of tumor immune-infiltrated cells have strong connection with METTL5. Furthermore, the upregulation of METTL5 was strongly correlated with the immune regulation of immunomodulators, chemokines, and chemokine receptors in the immune microenvironment. The oncogenesis and development of HCC are closely related to METTL5 expression, and the overexpression of METTL5 resulted in the poor survival outcome of HCC patients by regulating tumor immune microenvironment.


Results
Expression levels of METTL5 gene in HCC. We calculated the differential expression of the METTL5 gene in various types of cancers and matched normal tissues by means of online searching at the GEPIA database (Fig. 1A). The METTL5 gene was markably elevated among HCC specimens in comparison with surrounding healthy tissues ( Fig. 2A). Further analyses were adopted for assessing METTL5 gene expression in relation to tumor stage, and the METTL5 gene expression gradually increased among the advent of HCC progression via GEPIA (Fig. 2B). Such finding was consistent with the outcomes obtained from the Timer database (Fig. 1B). The aforementioned data implied that METTL5 gene overexpression may play a significant role in the tumorigenesis for HCC.

Potential prognostic effects of METTL5 gene expression on HCC.
We evaluated the prognostic significance of METTL5 expression on HCC patients using GEPIA. METTL5 gene overexpression is disadvantageous in terms of the overall survival time (OS) (Fig. 3A) and disease-free survival (DFS) (Fig. 3B) of HCC patients.
Expression profiles of METTL5 transcript in HCC. The available expression data of the METTL5 transcript in multiple HCC tissues were evaluated online from the UALCAN and HPA databases. Findings from both databases revealed that the intensified mRNA expression of METTL5 was observed in HCC specimens in comparison with adjacent healthy tissues via UALCAN (Fig. 4A) which is consistent with the outcomes of HPA database (Fig. 4B). Further analyses were used to assess the METTL5 transcript expression in relation to various clinical characteristics of HCC by using UALCAN. The results proved that METTL5 mRNA in HCC specimens was up-regulated in terms of age from 21 to 40, N1, stage 4, grade 4, Asian race, TP53 mutation, and male patients (Fig. 5).
Potential prognostic effect of METTL5 transcript expression on HCC. We further evaluated the impact of METTL5 mRNA expression on the survival outcomes of HCC patients using the KM database. The results showed that METTL5 mRNA overexpression had a close association with poor OS and PFS in HCC patients ( Fig. 6A and B). We also confirmed that increased METTL5 mRNA expression was strongly linked with   (Fig. 6C). These data implied that the overexpression of METTL5 mRNA in HCC predicted poor prognostic outcomes.
Expression profiles of METTL5 protein in HCC patients. METTL5 protein expression was substantially elevated in liver cancer when compared with paired normal tissues through the UALCAN database ( Fig. 7A and B). The upregulation of METTL5 protein was commonly detected in 21-40 year-olds and female patients via UALCAN ( Fig. 7C and D).
Potential prognostic effects of METTL5 protein expression on HCC patients. We further evaluate the impact of METTL5 protein expression on the survival outcomes of HCC patients using the HPA database. The high expression of METTL5 protein generally indicated the poor survival outcomes of HCC patients (Fig. 8A).
Genetic alteration of METTL5 in HCC patients. Hypermethylation of METTL5 gene was displayed in HCC in comparison with paired healthy specimens (Fig. 2C), inferring that the upregulation of METTL5 methylation contributed to METTL5 overexpression in HCC. Genetic alterations of METTL5 in HCC patients were analyzed using the cBioPortal tools. Our findings revealed that the genomic alterations of METTL5 occurred in 0.7% of HCC patients (Fig. 2D). All these results revealed that METTL5 genomic alteration indeed occur in HCC patients.
Gene enrichment analysis of METTL5 in HCC patients. A total of 8600 genes correlated with METTL5 were shown in the volcano plot via LinkedOmics (Fig. 9A), which comprised 3568 positively correlative genes and 5032 adversely correlative genes. The top 50 most prominent genes with positive and negative connections with METTL5 were displayed ( Fig. 9B and C). The METTL5-related enriched GO term and KEGG pathway were analyzed. Significant biological processes (BPs) indicated that METTL5-related genes were intimately engaged in translational initiation, protein localization to endoplasmic reticulum, mitochondrial gene expression, ribonucleoprotein complex biogenesis, and ncRNA processing (Fig. 10A). These interactive genes served as cellular components of ribosome, mitochondrial protein complex, cytosolic part, mitochondrial inner membrane, and respiratory chain (Fig. 10B). The molecular function enrichment of METTL5-related genes is mainly projected in ribosome, rRNA binding, electron transfer activity, unfolded protein binding, and oxidoreductase activity. Acting on a Heme group of donors with Heme-copper terminal oxidase activity (Fig. 10C), METTL5 were proven to largely participate in the signaling pathways of ribosome, oxidative phosphorylation, spliceosome, Parkinson disease, proteasome, and Huntington disease (Fig. 10D).  PPI network of METTL5. The PPI network was used to visualize the interaction between METTL5 and its relevant protein through the use of GeneMANIA tools. METTL5 was mainly enriched in the regulation of S-adenosylmethionine-dependent methyltransferase activity, methyltransferase activity, transferase activity, transferring one-carbon groups, methylation, RNA modification, and N-methyltransferase activity (Fig. 8B).   Correlation between METTL5 expression and chemokines in HCC. METTL5 expression presented a strongly positive association with several chemokines of CCL15, CCL16, CCL20, CXCL17, and XCL1 (Fig. 14A). Moreover, METTL5 expression was positively linked with the chemokine receptors of CCR4 and CX3CR1 (Fig. 14B). These findings indicated that METTL5 played a critical function in the immune interaction in HCC.

Discussion
HCC is a widely recognized prevalent malignant tumor with highly aggressive and fatal biological behavior across the world 10 . An increasing body of evidence revealed that exposure to chronic hepatitis virus infection 11 , persistent alcohol addiction 11 , and host tumor immune microenvironment 12 were principally responsible for the initiation and development of HCC. Surgical resection is a widely accepted optimal therapy for HCC patients at www.nature.com/scientificreports/ the earliest stages; however, even after the surgery of a patient, HCC is still accompanied by a high recurrence rate 13 . Although massive advancement was achieved in the aspect of therapeutic strategies in recent years, the clinical survival of HCC patients was far from satisfactory. Thus, promising diagnostic and prognostic biomarkers in HCC are needed to enhance the potential of precision treatments. Cancer cells are typically characterized by the malignant biological behavior of unrestrained cellular growth 14 . Recent studies reported that cancer cells exhibit the molecular characteristic of misregulated ribosome biogenesis 15 . m6A modification of rRNAs at the sixth adenine site at position 1832 of 18S rRNA (m6A1832) is emerging as a crucial oncogenic signal that facilitates carcinogenesis and rapid progression 16 . METTL5 was widely recognized as the methyltransferase that took charge of specifically catalyzing 18S rRNA m6A1832 modification 17 . A growing body of evidence displayed that the aberrant expression of METTL5 existed in a wide variety of human malignant tumors. A previous study reported that METTL5 is crucial for breast cancer cell growth 7 . Previous researchers clearly proposed that METTL5 expression considerably upregulated in HCC cells, targeted the knockdown of METTL5 suppressed proliferation and invasion abilities of HCC cells, and further induced cancer cell apoptosis in vitro tests 18 . Recent studies emphasized that METTL5 stimulated the proliferation and invasion activity of tumor cells in pancreatic cancer 19 . The study was conducted to evaluate the influence of METTL5 on the tumorigenesis and survival of HCC.
The correlation between METTL5 gene and HCC expressions was investigated using various databases. Our analysis exhibited that METTL5 gene expression was markably elevated in HCC. Furthermore, the METTL5 gene expression gradually increased with the development of the severity of liver cancer. Recent studies revealed that METTL5 functioned as an oncogene that produced a marked effect on the hyperactivation of tumor cell proliferation, migration, and invasion 19 . HCC patients had the worst survival in the presence of enhanced METTL5. Our study revealed that intensified mRNA expression of METTL5 were observed in HCC specimens in comparison with adjacent healthy tissues. METTL5 mRNA in HCC specimens was up-regulated in N1, stage 4, and grade 4 diseases. Our study reported that the overexpression of METTL5 mRNA had a close association with poor OS, PFS, and RFS among HCC patients. Increased METTL5 mRNA expression was strongly linked with unfavorable OS and PFS in stage 3, stage 3 + 4, and AJCC T3 diseases and male HCC patients. METTL5 protein expression was substantially elevated in liver cancer when compared with paired healthy tissues. The overexpression of the METTL5 protein had adverse effects on the prognosis of HCC. The trend of METTL5 expression in the aspects of transcript and protein was substantially in line with that of the gene, that is, METTL5 gene, transcript, and protein have the same influence on the prognosis of HCC. These observations strongly supported that METTL5 www.nature.com/scientificreports/ might be taken as a potential diagnostic and prognostic indicator for HCC patients. The final results exhibited that the hypermethylation of METTL5 indeed enhanced the expression of METTL5 in HCC samples. Related functional networks of METTL5 gene are significantly implicated in ribosome signaling, oxidative phosphorylation, spliceosome, proteasome, RNA transport, and mismatch repair, which were consistent with the previous findings that METTL5 alterations engaged in post-transcriptional regulation and ribosome biogenesis 20 . Existing literature reports indicated that METTL5 played an essential role in the promotion of translation initiation 7 , and previous studies revealed that METTL5 participated in the modulation of mismatch repair 21 .
Immune infiltration played a pivotal role in the carcinogenesis and rapid progression of HCC 22 . The infiltration of immune cells was an essential component of the immune microenvironment and contributed to the mediation of tumor proliferation and metastasis 23 . TIMER tools were further adopted to investigate the underlying correlation of immune infiltration and HCC. METTL5 expression showed positive correlation with the infiltration degrees of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophil, and dendritic cells in HCC. Accumulative evidence suggested that the interplay between neutrophils and circulating tumor cells could www.nature.com/scientificreports/ facilitate distant metastasis of tumor cells 24 , and macrophages were proven to enhance tumor cell migration and invasion by promoting the EMT process 25 . Our data revealed that METTL5 expression had an intimate link with the majority of the examined marker genes of B cells, CD8+ T cells, M1 macrophage, neutrophil, dendritic cell, Th1 cells, Th2 cells, and other known immune cells. Moreover, increased METTL5 expression showed a significant correlation with immunostimulators, immunoinhibitors, chemokines, and chemokine receptors. The aforementioned results proved the potential immune function of METTL5 in HCC, implying that its overexpression resulted in HCC by mediating the immune contexture. Numerous studies identified PD-1 as a principally expressed in CD4+ T cells, and other types of immunocyte and the high abundance of PD-1 contributed to the modulation of immune evasion 26,27 . The interaction of PD-1 and PD-L1 was regarded as a crucial mechanism for evading anti-tumor immunity 28 . CTLA-4 served as the immunoinhibitory molecule universally produced by highly activated Treg cells 29 . PD1/PDL1 and CTLA-4 were indispensable components of immune checkpoint inhibitors; PD1/PDL1 or CTLA-4 checkpoint blockade therapy exhibited potential therapeutic effects on HCC patients 30 . Our results suggested that METTL5 expression had a positive link with PD1 (PDCD1) and CTLA4 in HCC specimens. The aforementioned results reflected that METTL5 might be used as a promising target in antitumor immunotherapy.
Our study suggested that METTL5 expression in HCC specimens had a close link with kinase networks, such as myosin light chain kinase (MLCK), aurora kinase B (AURKB), ribosomal protein S6 kinase A4 (RPS6KA4) functioned on modulating mitosis, cell cycle checkpoint, DNA damage response, cell growth, and cell proliferation 31,32 . AURKB produced marked effects on tumorigenesis and genomic instability 31 . MLCK participates in the regulation of cellular processes for cell adhesion and migration 33 . MLCK was identified as a critical modulator of mitotic division 34 , and the decline in MLCK expression or MLCK activity inhibition highly attenuated the proliferation capacity of different kinds of cancerous cells 35,36 . RPS6KA4 was considered to play the cancer-promoting effects of HCC 32 .
MiRNAs refer to short noncoding RNAs (20-24 nucleotides) that serve as key molecular components of posttranscriptional regulation in controlling gene expressions to facilitate human oncogenic transformation 37 . Our The hyperactivation of MIR-127 regulates NF-κB signaling to inhibit HCC cell proliferation 40 . miR-423 was discovered to contribute to HCC progression by modulating the BP of cell growth and cell cycle 41 . MIR-510 also participated in tumor development 42 . The aforementioned results implied that the dysregulation of these miRNAs led to the initiation and progression of HCC. Several limitations existed in our study. Firstly, most of our data were principally extracted on the basis of online platform databases. Secondly, there were few data from public databases, which were utilized to analyze the relationship between chronic liver diseases and METTL5 expression, so we couldn't further investigated whether METTL5 expression contributed to HCC occurrence and progression by affecting the process of chronic liver diseases. Hence, further experimental validation must be conducted in subsequent studies. Finally, the assessment of "low" and "high" METTL5 expression was not elaborated in these online public databases, a more precise analysis should be conducted if a detailed and accurate cut-off level of METTL5 expression is set.  www.nature.com/scientificreports/ LinkedOmics. Various analyses of functional enrichment, kinase-target enrichment, and miRNA-target enrichment were conducted by analyzing cancer-associated datasets through the LinkedOmics websites (http:// linke domics. org/ admin. php) 48 . The meta p-value was analyzed by utilizing "RankCriteria", the "Minimum Number of Genes Size" was set as 3 and the "Simulations" was set as 500.
GeneMANIA. Protein-protein interaction (PPI) networks were constructed through the publicly available platform of GeneMANIA (http:// genem ania. org/) 50 . Interactive METTL5 genes derived from the Metascape dababase were inputted into the GeneMANIA for the assessment of gene functions.
TIMER. TIMER (https:// cistr ome. shiny apps. io/ timer/) was applied to systematically assess the correlation of METTL5 with immune cell infiltration in various malignancies and evaluate the impact of immune infiltration on survival outcomes 51 . The relationship between METTL5 expressions and the levels of immune cell infiltration in LIHC were assessed by utilizing the "Gene" module. The correlation between the copy number alterations of METTL5 and the levels of immune cell infiltration through the "SCNA" module.
Ethics approval and consent to participate. Ethics approval from the Institutional Review Board and written informed consent from eligible patients were not required because the data are publicly accessible and patient's private information was deleted.

Data availability
Publicly available datasets were analyzed during the present study. This data can be found here: http:// gepia. cancer-pku. cn/.